Human papillomavirus entry: late trafficking and establishment of infection

反式高尔基体网络(TGN)是HPV胞质转运的中间靶细胞器 有传染性的进入。以前已经证实，病毒基因组需要核膜破裂。 才能进入原子核。我们最近发现，病毒基因组在早期从TGN解离，并与 微管聚集在微管组织中心。在有丝分裂后期(中期)，病毒 基因组从有丝分裂的纺锤体极移向染色体。我们的数据指向 有丝分裂过程中微管介导的负端向正端转运的方向性。初步 抑制剂研究表明，Dynein和Kif11是参与转运的马达蛋白。方向性的转移和 目标1将进一步研究马达蛋白在微管介导的运输中的作用。而不是 病毒基因组从早期的胞内室中排出，仍然存在于膜结合区 整个有丝分裂运输的囊泡室。病毒基因组从膜小泡中流出 核包膜重塑。这些是范式转变的发现，最近得到了戴的证实 等人使用了电子显微镜。目前尚不清楚HPV是如何从核运输囊泡中逃脱的。我们 假设HPV利用细胞途径来溶解最终到达细胞核的小泡，无论是在 目的(如病毒感染)或意外。事实上，初步数据表明，特定的亚型 磷脂酶C在核传递后的某个阶段严重损害HPV16感染，可能是 参与了水泡膜的溶解。这一假设将在目标2中使用击倒和 基因敲除技术结合差异染色技术和EM。从转运囊泡中释放 在此之前是PML蛋白招募。招募PML核机构(Nb)的另一个组成部分Sp100， 在围绕输入基因组的PML NB组装中被特别延迟。在缺乏PML蛋白的细胞中，病毒 基因组丢失，因此转录不活跃。在目标2中，我们将检验我们的假设 特定的PML亚型是Sp100延迟招募的根本原因，HPV诱导的PML NB重组对于输入基因组的转录调控是重要的。我们最近描述了 一种新的细胞培养模型，可有效感染原代角质形成细胞以研究PML的作用 相关细胞培养模型中的NB组分。 总体而言，我们提出的研究将填补在理解微管介导的运输过程中的一个巨大空白 晚期HPV细胞内转运，将帮助我们解开PML NB在早期事件中的作用 对于确定感染至关重要的HPV生命周期。此外，该提案有可能揭示 一种到目前为止还未被识别的细胞途径，以溶解最终进入细胞核的小泡。即使是投机性的 在这个时候，这些观察可能会引发对囊泡向核运送货物的可能作用的重新思考。 有丝分裂期间及其在细胞核动态平衡中的作用。

The trans-golgi network (TGN) serves as intermediate target organelle of the cytoplasmic transport during HPV infectious entry. It has previously been established that nuclear envelope breakdown is required for viral genome to enter the nucleus. We recently found that viral genome dissociates from TGN in prophase and associates with microtubules to accumulate at the microtubule-organizing center. At later stages of mitosis (metaphase), viral genome moves away from the mitotic spindle poles towards chromosomes. Our data point to a switch in the directionality of microtubule mediated transport from minus end- to plus end-directed during mitosis. Preliminary inhibitor studies point to dynein and Kif11 as motor proteins involved in the transport. Shift in directionality and the role of motor proteins for microtubule mediated transport will be further studied in Aim 1. Rather than egressing from the endocytic compartment in prophase, viral genome is still found in a membrane-bound vesicular compartment throughout mitotic transport. Viral genome egresses from membrane vesicles after nuclear envelope reformation. These are paradigm shifting findings, which have recently been confirmed by Day et al using electron microscopy. It is unclear, how HPV achieves egress from the nuclear transport vesicles. We hypothesized that HPV utilizes a cellular pathway to dissolve vesicles ending up in the nucleus, whether on purpose (e.g. viral infection) or accidentally. Indeed, preliminary data suggest that specific isoforms of phospholipase C, whose knockdown severely impairs HPV16 infection at a stage after nuclear delivery, may be involved in dissolving the vesicular membrane. The hypothesis will be tested in Aim 2 using knockdown and knockout approaches combined with differential staining techniques and EM. Release from transport vesicles is preceded by PML protein recruitment. Recruitment of another component of PML nuclear bodies (NB), Sp100, is specifically delayed in PML NB assembling around incoming genome. In cells deficient for PML protein, viral genome is lost and therefore transcriptionally inactive. In Aim 2, we will test our hypothesis that the recruitment of specific PML isoforms is the underlying reason for delayed recruitment of Sp100 and that HPV-induced PML NB reorganization is important for transcriptional regulation of the incoming genome. We have recently described a novel cell culture model that allows efficient infection of primary keratinocytes for investigating the role of PML NB components in a relevant cell culture model. Overall, our proposed studies will fill a huge gap in the understanding of microtubule mediated transport during late HPV intracellular trafficking and will help us unravel the role of PML NB during the immediate early events of the HPV life cycle essential for establishing infection. Furthermore, the proposal has the potential to uncover a hitherto unrecognized cellular pathway to dissolve vesicles ending up in the nucleus. Even though speculative at this time, the observations may trigger a rethinking of possible roles of vesicular cargo delivery to the nucleus during mitosis and its role in nucleus homeostasis.